Method for plant regeneration of okra

ABSTRACT

The present description concerns methods for regeneration of whole plant from the explants obtained from the  Abelmoschus  species preferably  A. esculentus . In addition the present description also concerns methods for transforming okra plant, plant cells and tissues either with the use of recombinant  Agrobacterium  strain or by bombarding the explants with tungsten or gold particles coated with DNA sequences of interest. An efficient method to isolate embryos from imbibed seeds of okra is also described which enables the use of young meristematic cells of plumule tip for efficient regeneration and transformation of okra plants. Further, transformed okra plants, plant cells and tissues for improved agronomic/non agronomic traits and insect resistance are produced either by using marker based or marker free systems.

RELATED APPLICATIONS

The present application is a Divisional of U.S. application Ser. No.11/574,007, which is a National Phase entry of International ApplicationNumber PCT/IN2005/000277, filed Aug. 16, 2005, which claims priorityfrom Indian Application No. 1522/DEL/2004, filed Aug. 20, 2004, thedisclosures of which are hereby incorporated by reference herein intheir entirety.

1. TECHNICAL FIELD

The present invention provides novel methods of regeneration of wholeplant from explants of okra plant and other Abelmoschus species.Further, the invention also provides methods for transformation ofplant, plant cells and tissues of Abelmoschus species either using theAgrobacterium mediated method or the particle bombardment method.Transgenic insect resistant okra plants were generated, either by usingmarker based systems or marker-free systems.

2. BACKGROUND AND PRIOR ART REFERENCES 2. A. Okra

Okra (Abelmoschus esculentus) is one of the most important vegetablecrops. The fruits are consumed in various forms in a number ofcountries. Okra has also been used as a source of fiber and for theproduction of oil and proteins.

Okra is susceptible to many insect pests and diseases which reduce theyield across the Okra growing regions. Okra yellow vein mosaic virus isa devastating disease in India and many other countries. This crop isextensively damaged by the Lepidopteran insect/pests viz.; Shoot andfruit borer (Earias vitella, E. insulana) and the Fruit borer(Helicoverpa armigera). Genetic improvement by conventional plantbreeding is impaired due to the lack of resistance sources to pests anddiseases in Okra germplasm.

2. B. Plant Cell and Tissue Culture

Each plant cell has the inherent ability for independent developmentinto a whole organism if provided with the proper external conditions.Since the early demonstration of this ability, viz., totipotency anddifferentiation in vitro, plant tissue culture techniques have beenwidely used in the clonal multiplication of plants (Herberlandt Sber.Akad. Wiss. Wien. (1902) 111:69-92).

Plant tissue culture technology is making significant contributions toagriculture in the clonal propagation, haploid breeding, mutantcultures, pathogen free plants, cryopreservation of plant tissues forthe establishment of in vitro gene banks, production of secondaryproducts and genetic engineering of plants (Chilton, Scientific American(1983) 248.6:36-45).

The prospects of success with the genetic engineering of plants havecreated considerable public interest. This technique involves theinsertion of foreign genes into plant cells using vectors and theregeneration of whole plants from transformed single cells using planttissue culture techniques. Although tissue culture based plantregeneration methods have been standardized for a wide variety of plantspecies, many crops have been recalcitrant and thus restricts thegenetic engineering potential of these plants.

2. C. Tissue Culture of Okra

Tissue culture based direct plant regeneration of okra has beendescribed by Mangat and Roy., Plant Science (1986) 47:57-62. Thisinvestigation outlines the comparative tissue culture responses ofhypocotyl, cotyledon, cotyledonary node and primary leaf explantsaseptically grown okra seedlings cultured on 6 different media. Theplant regeneration was not attained from hypocotyl and leaf segmentexplants in all the 6 media tested. The cotyledon explants respondedmoderately in plant regeneration, on one out of 6 media tested.Cotyledon node explants did not regenerate on 3 of the media, whereas,responded low in shoot regeneration on 1 medium, and responded very highin the shoot regeneration on another medium.

In the above publication the shoot regeneration frequency is mentionedas nil, low, moderate and very high and not in figures (%). It makes thecomparison of regeneration frequency, difficult.

In a second publication, Roy and Mangat (Roy et al., Plant Science(1989) 60:77-82) have reported the regeneration of plants fromcotyledonary-axil derived callus tissue of Okra. Callus induction thatresulted from all the explants cultured on MS medium supplemented withthe benzyl adenine (BA). The hypocotyl-derived callus remainednon-organogenic, whereas, cotyledonary-axil-derived callus producedshoots. The addition of silver nitrate in media resulted in up to 74% ofthe bud primordia going onto produce shoots.

The study of prior art shows no method available for the regeneration ofplants from plumule of embryo. Our present inventions describe methodsfor the high efficiency plant regeneration from plumule of okra for thefirst time.

The study of prior art also shows no method available for theregeneration of plants from the other explants also tested during in ourinvestigation.

2. D. Plant Transformation and Generation of Transgenic Plants

The development of gene transfer techniques for plant species is ofgreat interest, importance and value because it can be used for thetransfer of beneficial genes of interest into plants.

A variety of techniques have been used to introduce foreign genes intoplant cells. Agrobacterium mediated transformation has been described byMurai et al., Science (1983) 222:476-482, Fraley et al., Proc. Natl.Acad. Sci. USA (1983) 80:4803-4807; Direct DNA uptake method has beendescribed by Lorz et al., Mol. Gen. Genet., (1985) 199:178-182,Portrykus et al., Mol. Gen. Genet., (1985) 199:183-188; Microinjectionmethod has been described by Crossway et al., Mol. Gen. Genet., (1986)202:179-185; High velocity micro-projectile method has been described byKlein et al., Nature (1987) 327:70-73 and Electroporation method hasbeen described by Fromm et al., Proc. Natl. Acad. Sci. USA (1985)82:5824-5828, Fromm et al., Nature (1986) 319:791-793.

2. E. Agrobacterium-Mediated Transformation

One of the most common methods of introducing foreign genes into plantcells is through Agrobacterium-mediated transformation. Agrobacterium isa natural plant pathogen and it mediates genetic transformation as partof the natural process it utilizes when it infects a plant cell. Duringthe process of transformation a specific segment of the vector which isknown as T-DNA, is transferred into the cells. The T-DNA ofAgrobacterium can be engineered to contain gene/s or DNA sequences ofinterest that can be transferred into the host plant cells and insertedinto the plant genome.

Agrobacterium-mediated transformation is attractive because of the easeof the protocol coupled with minimal equipment costs. Moreover,transgenic plants obtained by this method often contain a single copy ofT-DNA integrations.

Agrobacterium-mediated transformation and the subsequent regeneration oftransgenic plants carrying inserted genes were described by Murai etal., Science (1983) 222:476-482. Fraley et al., Proc. Natl. Acad. Sci.USA (1983) 80:4803-4807. De Block et al., The EMBO Journal (1984)3:1681-1689 and Horsch et al., Science (1985) 227:1229-1231.

2. F. Biolistic-Mediated or Particle Bombardment Method ofTransformation

Another common method of introducing foreign gene/s into plant cells isusing particle bombardment which is also known as biolistic or highvelocity microprojectile. The basis of particle bombardment is theacceleration of particles coated with gene/s of interest toward cells,resulting in the penetration of the protoplasm by the particles andsubsequent expression of the introduced gene/s. In this method heliumpressure is used to accelerate particles coated with DNA into the cells.

Microprojectile bombardment can transform diverse target tissues.Particle bombardment and subsequent regeneration of transgenic plantscarrying inserted genes were described by Klein et al., Nature (1987)327:70-73. Klein et al., Bio/Technology (1992) 10:286-292. Casas et al.,Proc. Natl. Acad. Sci. USA (1993) 90: 11212-11216.

2. G. Transformation of Okra

The study of prior art shows no method available for the transformationof Okra plant, cells and tissues. Our present inventions describemethods for the transient and stable transformation of okra plant, cellsand tissues using Agrobacterium-mediated and biolistic transformationsystems for the first time.

2. H. Marker Based Transformation Systems

In the marker based transformation system, the gene of interest and theselectable marker gene (Eg. NPT II gene) are linked. In the marker basedAgrobacterium-mediated transformation system, the T-DNA is engineered tocontain the gene of interest and the marker gene. Where as in the markerbased biolistic transformation system the plasmid used, contains thegene of interest and the selectable marker gene. The transgenic plantsgenerated from the above systems contain the marker gene along with thegene of interest.

2. I. Marker-Free Transformation Systems

Marker-free transformation systems have the advantages of introducingagronomical important genes, and at the same time, avoiding theintroduction of the selectable marker genes. Different methods have beenemployed for the generation of marker-free transgenic plants. Thesemethods include Agrobacterium-mediated co-transformation, excision ofthe selectable marker via cre/lox recombination, use of transposableelements, co-bombardment of the plasmids and altered metabolism (Yoderet al., Bio/Technology (1994) 12:263-267).

Agrobacterium-mediated co-transformation, using two separate plasmids ina single Agrobacterium, i.e. one vector carrying the selectable markergene in one T-DNA and the other vector carrying the gene of interest inanother T-DNA are used. The transgenic plants generated from thistransformation system are analyzed and the plants having gene ofinterest but with out selectable marker gene are selected in the furthergenerations from the segregated progenies (Komori et al., The PlantJournal (1996) 10: 165-174).

2. J. Engineering Plants for Insect Resistance

2. J. A. Bacillus Thuringiensis (Bt) Gene

Bacillus thuringiensis (Bt) is a gram positive bacterium which producesa variety of insecticidal crystal proteins toxic to insects. These Btgenes have been successfully engineered into crop plants to getresistance to the specific insect pests in a number of crops. Forexample insect resistant transgenic tomato plants were generated with Btgene by Fischhoff et al., Bio/Technology (1987) 5:807-813.

2. J. B. Protease Inhibitors

Protease inhibitors are an important element of plant defense responseto insect predation. Transgenic plants expressing protease inhibitorsshow enhanced resistance to predation by pests, indicating the usefulfunction of these inhibitors (Johnson et al., PNAS (1990) 86:9871-9875.

2. K. Insect Bioassays

The bioassays using specific insect pests with plants/plant parts areconducted to understand the resistance or susceptibility of the planttowards the pest. The efficacy of the specific insect resistant proteinexpressed in transgenic plants is tested with the specific target pestin the insect bioassays. The resistance of the plant towards specifictarget pests is compared with non-transgenic plant controls.

3. OBJECTS OF THE INVENTION

An object of the present invention is to provide methods forregeneration of okra plant and Abelmoschus species.

Another object of the present invention is to provide methods fortransformation of okra plant, plant cells and tissues.

Still another object of the present invention is to provide methods totransform okra plant, plant cells and tissues by co-cultivating theexplants with recombinant Agrobacterium strain comprising DNA/RNAsequences of interest.

Still another object of the present invention is to provide methods totransform okra plant, plant cells and tissues by bombarding the explantswith tungsten or gold particles coated with DNA sequence of interest.

Further, another object of the present invention is to obtain okraplant, plant cells and tissues which carry and/or confer the traits ofagronomic and non-agronomic importance.

Still another object of the present invention is to obtain okra plant,plant cells and tissues which confer tolerance or resistance to disease,herbicide or insects.

Still another object of the present invention is to obtain marker-free,transgenic, okra plants.

Still another object of the present invention is to obtain marker-free,transgenic insect resistant okra plants.

4. SUMMARY OF THE INVENTION

The present invention provides novel and efficient methods ofregeneration of whole plant of Abelmoschus species preferably A.esculentus.

Another aspect of the present invention is to provide methods forregeneration of whole plant of Abelmoschus species wherein the explantsare selected from a group consisting of cotyledon with petiole,hypocotyls, embryo, immature embryo, leaf lamina, cotyledonary axil,shoot tip, anther, root, callus or other suitable explants.

The invention provides for a method of regeneration of the explantswherein the explants are cultured on a regeneration medium (MS0Z₂medium) containing a cytokinin wherein the cytokinin is selected from agroup consisting of zeatin, BAP, kinetin, TDZ, Adenine sulphate, Adeninefree-base alone or in combination, preferably Zeatin, wherein theconcentration of zeatin is in the range 0.01 to 5 mg/l, preferably 2mg/l. The explant can also be regenerated on other media known in theart having a combination of cytokinins in the range of 0.01 to 20 mg/lor combination of cytokinins and auxin, with the latter in the range of0.01 to 5 mg/l.

Further, the explants are incubated at a temperature of 18° C. to 30°C., preferably 26° C. and luminosity of 250 to 5000 lux for theregeneration of multiple shoot buds. The invention also provides forfurther multiplication of shoot buds on the medium as described above.

The invention also provides a method wherein the multiplied shoot budsare further transferred to a medium without growth regulator (MS0medium) or medium with low concentration of auxin such as NAA, 2,4-D andIAA and cytokinin such as zeatin, BAP, kinetin, Adenine in the range of0.01 to 2 mg/l for further elongation of shoot and the induction andgrowth of roots to obtain plantlets.

In another aspect of the invention, the plantlet obtained isphenotypically normal and fertile and is capable of producing thefertile seeds in subsequent generations.

In another aspect of the invention, the plantlet obtained is a mutantand fertile and is capable of producing the fertile seeds in subsequentgenerations

The present invention also provides a novel and efficient methods fortransforming plant, plant cells and tissues of Abelmoschus species usingAgrobacterium-mediated or particle bombardment methods.

Different varieties or accessions of Abelmoschus esculentus species areselected for transforming plant, plant cells and tissues usingAgrobacterium-mediated or particle bombardment methods.

In another aspect of the invention the explants for transformation areselected from a group consisting of cotyledon with petiole, hypocotyls,embryo, immature embryo, leaf lamina, cotyledonary axil, shoot tip,anther, root and callus or any other suitable explants.

Another aspect of the present invention is to devise an efficient methodto isolate embryos from imbibed seeds of okra which enables the use ofyoung meristemic cells of plumule end for transformation.

Another aspect of this invention is to provide a method of wounding toincrease transformation efficiency. The wounding involved pricking orpenetrating the embryo and other explants by a sharp object, a needle oran abrasive object.

Further the transformed explants are cultured on medium containing acytokinin, wherein the cytokinin is selected from a group consisting ofzeatin, BAP, kinetin, TDZ, Adenine sulphate, Adenine free-base alone orin combination, preferably Zeatin, wherein the concentration of zeatinis in the range 0.01 to 5 mg/l, preferably 2 mg/l (MS0Z₂H₁₀C medium) ora combination of cytokinins in the range of 0.01 to 20 mg/l orcombination of cytokinins and auxin, with the latter in the range of0.01 to 5 mg/l, containing antibiotic hygromycin in the range of 5 mg/lto 100 mg/l, preferably 10 mg/l for selection of transformed plant cellsand tissues and for the generation of multiple shoot buds.

Further the transformed and multiplied shoot buds are transferred onshoot elongation media as described above and then in another media withlow concentration of auxin such as NAA, 2,4-D and IAA and/or cytokininsuch as zeatin, BAP, kinetin, Adenine or MS basal medium for inductionand growth of roots to obtain whole plants.

Another aspect of the present invention is to provide a methods fortransforming plant, plant cells and tissues of Abelmoschus species usingAgrobacterium-mediated or particle bombardment methods wherein thetransformed okra plant carry the DNA/RNA sequence of interest whereinthe transformed plant shows improved agronomic traits or a combinationof traits comprising for yield, drought resistance, stress resistance,nutritional value, inducing male sterility into the plant, cells andtissues.

Another aspect of the present invention is to provide methods fortransforming plant, plant cells and tissues of Abelmoschus species usingAgrobacterium-mediated or particle bombardment methods wherein thetransformed okra plant carry the DNA/RNA sequence of interest whereinthe transformed plant shows tolerance or resistance to disease,herbicide or insects.

Another aspect of the present invention is to provide a method ofgenerating marker-free, transgenic okra plants, with a nucleotidesequence of interest.

Another aspect of the present invention is to provide transformed okraplants showing tolerance or resistance to disease, herbicide or insects

5. BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS

FIG. 1: Transformed mature embryo, cotyledonary axil, shoot tip andcallus by Agrobacterium-mediated method showing GUS activity.

FIG. 2: Transformed mature embryo, callus and by anther Biolistic-methodshowing GUS activity.

FIG. 3: A) Multiple shoot buds developed from the plumule tip of theembryo. B) Elongated shoots and Rooting of regenerated okra plant. C)Hardened okra plant in green house showing fruiting

FIG. 4: Transformed seedlings of T1 generation showing GUS activity.

6. DETAILED DESCRIPTION OF THE INVENTIONS

An embodiment of the present invention is to provide a method ofregenerating whole plant from explants of Abelmoschus esculentusspecies, wherein the said method comprising the steps of:

-   -   a) surface sterilizing the seeds and imbibing the seeds in water    -   b) germinating the surface sterilized seeds of step (a) on a        suitable culture media to obtain seedlings,    -   c) obtaining the explants from step (a) or step (b),    -   d) wounding the explants from step (c),    -   e) culturing the explants of step (c), on a suitable medium to        obtain multiple shoot buds or callus    -   f) culturing the shoot buds of step (e) on a suitable medium for        further elongation of shoots and induction of roots to obtain        rooted plantlets    -   g) Transferring the rooted plantlets of step (f) in soil, to        obtain fertile R₀ plants,    -   h) Advancing the plants of step (g) to subsequent generations.

Further, the invention provides for regeneration of plants fromAbelmoschus species wherein the species are selected from a groupconsisting of A. caillei, A. moschatus, A. manihot, A. tuberculatus, A.ficulneus, A. crinitus, A. angulosus A. tetraphyllus.

Another embodiment of the present invention provides a method ofregeneration of okra plant, wherein the surface sterilized seeds arewashed and imbibed for a period of 0 to 48 hours, and then placed on MS0medium for germination [MS0 medium contains MS salts, B5 vitamins, 0.8%agar, 3% sucrose and pH 5.8]

Further, the invention provides for use of various explants forregeneration of okra plant wherein the explants are selected from agroup consisting of cotyledon with petiole, hypocotyls, embryo, immatureembryo, leaf lamina, cotyledonary axil, shoot tip, anther, root, callusor other suitable explants.

Still another embodiment of the present invention is to provide a methodof regeneration of okra plant wherein the surface sterilized and imbibedseeds are used to isolate the embryos in sterile conditions by pressingwith tweezers or any other means and these embryos are washed in sterilewater and blotted dry on filter paper and later placed on soaked sterilefilter paper.

Another embodiment of the present invention is to provide a method ofregeneration of okra plant wherein the explants are transferred onto amedium containing Zeatin in the range 0.01 to 5 mg/l, preferably 2 mg/l(MS0Z₂ medium). The explants are placed for incubation in plant tissueculture incubation room at a temperature 18° C. to 30° C. (preferably26° C.) and luminosity of 250 to 5000 lux for the regeneration ofmultiple shoot buds. The explants may also be regenerated using othermedia compositions known in the art such as media containing acombination of cytokinins in the range of 0.01 to 20 mg/l or combinationof cytokinins and auxin, with the latter in the range of 0.01 to 5 mg/l.

Another embodiment of the present invention is to provide a method ofregeneration of okra plant wherein the multiple shoot buds regeneratedare excised and transferred onto a medium containing a cytokinin,wherein the cytokinin is selected from a group consisting of zeatin,BAP, kinetin, TDZ, Adenine sulphate, Adenine free-base alone or incombination, preferably Zeatin, wherein the concentration of zeatin isin the range 0.01 to 5 mg/l, preferably 2 mg/l (MS0Z₂ medium) or acombination of cytokinins in the range of 0.01 to 20 mg/l or combinationof cytokinins and auxin, with the latter in the range of 0.01 to 5 mg/l,and kept for incubation for the further multiplication of shoot buds.

Another embodiment of the present invention is to provide a method ofregeneration of okra plant wherein the said shoot is transferred ontomedium and allowed to grow to a length of 0.5 cm to 7 cm, preferably 1.5cm.

Another embodiment of the present invention is to provide a method ofregeneration of okra plant wherein, the elongated shoot is transferredto a medium without growth regulator (MS0 medium) or medium with lowconcentration of auxin such as NAA, 2,4-D and IAA and/or cytokinin suchas zeatin, BAP, kinetin, Adenine in the range of 0.01 to 2 mg/l forfurther elongation of shoot and the induction and growth of roots toobtain plantlets.

Another embodiment of the present invention is to provide a method ofregeneration of okra plant wherein the plantlet is transferred in soilfor further growth. The regenerated plantlet grown is phenotypicallynormal and/or mutant and fertile and is capable of producing fertileseeds in subsequent generations. Further the invention provides foradvancing the generation of the regenerated Okra plants.

Still another embodiment of the present invention is to provide a methodfor transforming Abelmoschus species, wherein the said method comprisingthe steps of

-   -   a) surface sterilizing the seeds, and imbibing the seeds with        water,    -   b) germinating the seeds of step (a) in a suitable culture        medium to obtain seedlings,    -   c) obtaining the explants from step (a) or step (b),    -   d) wounding the explants of step (c),    -   e) co-cultivating the explants of step (d) with recombinant        Agrobacterium strain,    -   f) culturing the explants of step (e) on a suitable tissue        culture medium to select transformed plant cells and tissues,    -   g) culturing the transformed plant cells and tissues of step (f)        on a suitable tissue culture medium to obtain shoot buds, and    -   h) culturing the shoot buds of step (g) in a suitable rooting        medium to obtain rooted transformed plants.

Further, the invention provides for regeneration of plants fromAbelmoschus species wherein the species are selected from a groupconsisting of A. caillei, A. moschatus, A. manihot, A. tuberculatus, A.ficulneus, A. crinitus, A. angulosus A. tetraphyllus.

Another embodiment of the present invention is to provide a methodwherein the surface sterilized seeds are washed and imbibed in water fora period of 0 to 48 hours. Further, the surface sterilized seeds areplaced on MS0 medium for germination (MS0 medium contains MS salts, B5vitamins, 0.8% agar, 3% sucrose and pH 5.8)

Still another embodiment of the present invention is to provide a methodfor transforming Abelmoschus esculentus species, wherein the surfacesterilized seeds are used to isolate the embryos in sterile conditionsby pressing with tweezers or any other means and these embryos arewashed in sterile water and blotted dry on filter paper and later placedon soaked sterile filter paper.

Another embodiment of the present invention is to provide a method fortransforming Abelmoschus species, wherein the explants are selected froma group consisting of cotyledon with petiole, hypocotyls, embryo,immature embryo, leaf lamina, cotyledonary axil, shoot tip, anther, rootand callus or other suitable explants.

Another embodiment of the present invention is to provide a methodwherein the embryos or various explants of Abelmoschus species arewounded with a sterile needle or with any suitable sharp object.

Another embodiment of the present invention is to provide a method fortransforming Abelmoschus species, wherein the recombinant Agrobacteriumstrain carrying DNA/RNA sequence comprises of a coding or non-codinggene sequence, inclusive or not, of terminator or promoter, as anexpressing or non-expressing cassette.

Further, the Agrobacterium strain carrying DNA/RNA sequences confersimproved agronomic traits or combination of traits comprising for yield,drought resistance, stress resistance, nutritional value or inducingmale sterility into the plant, cells and tissues.

Another embodiment of the present invention is to provide methods fortransforming Abelmoschus species, wherein the DNA/RNA sequences conferstolerance or resistance to disease, herbicide or insects to transformedplant, cells and tissues.

Another embodiment of the present invention is to provide a method fortransforming Abelmoschus species, wherein the DNA/RNA sequences areeither coding or non coding sequences.

Another embodiment of the present invention is to provide methods forthe transformation of okra using marker-free systems.

Further, the explant is inoculated with the recombinant Agrobacteriumstrain containing DNA sequence of interest. The explant is blotted dryon sterile filter paper and transferred onto co-cultivation medium.Various media combinations may be used for co-cultivation medium whichis selected from a group consisting of MS0 medium or MSOAs medium orMS0Z₂ medium or MSOZ₂As medium.

The said medium MSOZ₂ or MS0Z₂As contains a cytokinin, preferablyZeatin, in the range 0.01 to 5 mg/l, preferably 2 mg/l (MS0Z₂ medium).

Further, the explant after co-cultivation is washed with liquid MS0medium with 500 mg/l Cefotaxime or suitable antibiotic to killAgrobacterium.

Another embodiment of the present invention is to provide a method fortransforming Abelmoschus species, wherein the said explant is blotteddry on sterile filter paper and can be cultured on a various media knownin prior art as medium containing a cytokinin, preferably Zeatin, in therange 0.01 to 5 mg/l, preferably 2 mg/l ((MS0Z₂H₁₀C medium) or acombination of cytokinins in the range of 0.01 to 20 mg/l or combinationof cytokinins and auxin, with the latter in the range of 0.01 to 5 mg/l,containing antibiotic for selection of transformed plant cells andtissues. Further the said explant is incubated in plant tissue cultureincubation room with temperature 18° C. to 30° C. and luminosity of 250to 5000 lux. The antibiotics used in media for selection of transformedplant cells and tissues can be either kanamycin in the range of 25 mg/lto 200 mg/l, preferably 50 mg/l or hygromycin in the range of 5 mg/l to100 mg/l, preferably 10 mg/l.

Further the said transformed plant cells and tissues is sub-cultured onmedium containing a cytokinin preferably Zeatin in the range 0.01 to 5mg/l, preferably 2 mg/l ((MS0Z₂H₁₀C medium) or a combination ofcytokinins or combination of cytokinins in the range of 0.01 to 20 mg/lor combination of cytokinins and auxin, with the latter in the range of0.01 to 5 mg/l, for further multiplication of shoot buds. The subcultured shoot buds were transferred onto MS0H₁₀C medium and allowed togrow to a length of 0.5 cm to 7 cm, preferably 1.5 cm.

Further the elongated shoot buds are transferred to a medium with lowconcentration of an auxin in the range of 0.01 to 2 mg/l for inductionand growth of roots.

Another embodiment of the present invention is to provide methods fortransforming Abelmoschus species, wherein the transformed shoot, plantcells and or tissues contain DNA/RNA sequences of interest, which iscoding for genes or not wherein the said DNA/RNA sequences aretransferred to subsequent generations by plant breeding techniquesincluding but not limited to crosses. Further the invention provides foradvancing the generation, wherein the said succeeding generationscontain DNA/RNA sequences of interest with or with out the selectablemarker gene (marker based or marker-free).

Another embodiment of the present invention is to provide a method forproducing transformed plant, plant cells and tissues of Abelmoschusesculentus species wherein the said method comprising the steps of

-   -   a) surface sterilization of seeds and imbibing the seeds in        water    -   b) germinating the seeds of step (a) in a suitable culture        medium to obtain seedlings or explants    -   c) obtaining the explants from seedlings of step (a) or (b)    -   d) bombarding the explants of step (c) with tungsten or gold        particles coated with DNA sequence of interest    -   e) culturing the explants of step (d) in a suitable culture        medium to select the transformed plant cells and tissues    -   f) maintaining the transformed plant cells and tissues of        step (e) in a suitable tissue culture medium to obtain shoot        buds    -   g) culturing the shoot buds of step (f) in a suitable rooting        medium for rooting to obtain transformed plants

Further, the invention provides for regeneration of plants fromAbelmoschus species wherein the species are selected from a groupconsisting of A. caillei, A. moschatus, A. manihot, A. tuberculatus, A.ficulneus, A. crinitus, A. angulosus A. tetraphyllus.

Another embodiment of the present invention is to provide a methodwherein the surface sterilized seeds are washed and imbibed in water fora period of 0 to 48 hours. Further, the surface sterilized seeds areplaced on MS0 medium for germination (MS0 medium contains MS salts, B5vitamins, 0.8% agar, 3% sucrose and pH 5.8).

Still another embodiment of the present invention is to provide a methodfor transforming Abelmoschus species, wherein the surface sterilizedseeds are used to isolate the embryos in sterile conditions by pressingwith tweezers or any other means and these embryos are washed in sterilewater and blotted dry on filter paper and later placed on soaked sterilefilter paper.

Another embodiment of the present invention is to provide a method fortransforming Abelmoschus species, wherein the explants are selected froma group consisting of cotyledon with petiole, hypocotyls, embryo,immature embryo, leaf lamina, cotyledonary axil, shoot tip, anther, rootand callus or other suitable explants.

Another embodiment of the present invention is to provide a method fortransforming Abelmoschus species, wherein the embryos and various otherexplants are wounded with a sterile needle and further bombarding theseexplants with tungsten or gold particles coated with 1 to 10 μg is ofDNA sequence of interest wherein the bombardment pressure is in therange of 600 to 1500 psi (preferably 1100 psi).

Further the bombarded explants can be cultured on a various media knownin prior art as medium containing a cytokinin, preferably Zeatin, in therange 0.01 to 5 mg/l, preferably 2 mg/l ((MS0Z₂H₁₀C medium) or acombination of cytokinins in the range of 0.01 to 20 mg/l or combinationof cytokinins and auxin, with the latter in the range of 0.01 to 5 mg/l,containing antibiotic for selection of transformed plant cells andtissues. Further the said explant is incubated in plant tissue cultureincubation room with temperature 18° C. to 30° C. and luminosity of 250to 5000 lux. The antibiotics used in media for selection of transformedplant cells and tissues can be either kanamycin in the range of 25 mg/lto 200 mg/l, preferably 50 mg/l or hygromycin in the range of 5 mg/l to100 mg/l, preferably 10 mg/l.

Further the said transformed plant cells and tissues is sub-cultured onmedium containing a cytokinin preferably Zeatin in the range 0.01 to 5mg/l, preferably 2 mg/l ((MS0Z₂H₁₀C medium) or a combination ofcytokinins or combination of cytokinins in the range of 0.01 to 20 mg/lor combination of cytokinins and auxin, with the latter in the range of0.01 to 5 mg/l, for further multiplication of shoot buds. The subcultured shoot buds were transferred onto MS0H₁₀C medium and allowed togrow to a length of 0.5 cm to 7 cm, preferably 1.5 cm.

Further the elongated shoot buds are transferred to a medium with lowconcentration of an auxin in the range of 0.01 to 2 mg/l for inductionand growth of roots.

Another embodiment of the present invention is to provide a method fortransforming Abelmoschus species wherein the said shoot, plant cellsand/or tissues contain DNA/RNA sequences of interest which confersimproved agronomic traits or combination of traits comprising for yield,drought resistance, stress resistance, nutritional value, inducing malesterility into the plant, cells and tissues or the transformed plant.

Another embodiment of the present invention is to provide a method fortransforming Abelmoschus species wherein the said shoot, plant cellsand/or tissues contain DNA/RNA sequences of interest which conferstolerance or resistance to disease, herbicide or insects to plant, cellsand tissues wherein the said DNA/RNA sequences coding for genes or not.

Further the said DNA/RNA sequences are transferred to subsequentgenerations by plant breeding techniques including but not limited tocrosses. Further the invention provides for advancing the generation,wherein the said succeeding generations contain DNA/RNA sequences ofinterest with or with out the selectable marker gene (marker based ormarker-free).

Still another embodiment of the present invention is to providetransformed Okra Plant, plant cells and tissues which carry traits ofagronomic and non-agronomic importance.

Still another embodiment of the present invention is to providetransformed plant, plant cells and tissues of Abelmoschus species usingAgrobacterium-mediated or particle bombardment methods wherein thetransformed okra plant carry the DNA/RNA sequence of interest whereinthe transformed plant shows improved agronomic traits or a combinationof traits comprising for yield, drought resistance, stress resistance,nutritional value, inducing male sterility into the plant, cells andtissues.

Still another embodiment of the present invention is to providetransformed plant, plant cells and tissues of Abelmoschus species usingAgrobacterium-mediated or particle bombardment methods wherein thetransformed okra plant carry the DNA/RNA sequence of interest whereinthe transformed plant shows tolerance or resistance to disease,herbicide or insects.

6. A. Regeneration of Abelmoschus Species by Tissue Culture Method

Okra (Abelmoschus esculentus) genotypes representing a variety ofagronomic varieties or hybrids [including Arka Anamika, Parbhani Krantiand proprietary lines of Maharashtra Hybrid Seeds Company Ltd. (MHSCL)]were used in these experiments. The seeds were surface sterilizedpreferably in 0.1% (weight/volume) HgCl₂ in distilled water for 2 to 60minutes, preferably 30 minutes (commercially available other surfacesterilizing agents also work instead of HgCl₂). These seeds were washedmany times in sterile distilled water. The sterilized seeds were imbibedwith water for a period of 0 to 48 hours. The seeds were germinated toobtain seedlings. Various explants were obtained from seeds andseedlings for regeneration of whole plants. The explants used for thepresent invention are selected from a group consisting of cotyledon withpetiole, hypocotyls, embryo, immature embryo, leaf lamina, cotyledonaryaxil, shoot tip, anther, root and callus or other suitable explants.These explants were used for regeneration of the whole plant.

6. A. 1. Cotyledon with Petiole

The seeds mentioned in 6.A. were inoculated on MS0 medium (MS0 mediumcontains MS salts, B5 vitamins, 3% sucrose, 0.8% agar and adjusted thepH 5.8) in bottles and incubated under light for the germination ofseeds in the tissue culture incubation room.

The explants viz., cotyledon with petiole were excised from seedlings (1to 20 days old preferably 12 days old seedlings). These explants wereinoculated on MS0Z₂ medium (MS0Z₂ medium contains MS salts, B5 vitamins,3% sucrose, 0.8% agar, with a cytokinin preferably 2 mg/l Zeatin andadjusted the pH to 5.8) and incubated in the tissue culture incubationroom.

Multiple shoot buds and calli were developed in more than 80% of theexplants, from the cut ends of the petiole of cotyledon explants withpetiole. Upon further transfer of the multiple shoot buds up to 5 foldmultiplication was obtained. These shoot buds were separated andsubcultured on MS0 medium for the further elongation of the shoots andinduction of rooting. In 2 to 3 weeks these shoots elongated further andthe roots become well developed. The rooted plants were hardened andestablished in green house. The procedures for hardening are similar tothose in prior art.

All the tissue cultured plants grew normally till maturity. Seed settingwas normal in all the plants.

The calli produced from cut ends of the petiole and lamina of cotyledonswere subcultured at an interval of 2 to 4 weeks (preferably 3 weeks) onMS0Z₂ medium. By the end of this period these calli developed to alarger size.

6. A. 2. Hypocotyls

The seeds mentioned in 4.A. were inoculated on MS0 medium in bottles andincubated under light for the germination of seeds. The hypocotylexplants were excised from 5 to 20 days old seedlings (preferably 12days old seedlings). These explants were inoculated on MS0Z2 medium.

Multiple shoot buds and callus were developed in 75+% of the explants,from the cut ends of hypocotyl explants in 2 to 6 weeks period. Uponfurther transfer of the multiple shoot buds up to 5 fold multiplicationwas obtained. These shoot buds were separated and subcultured on MS0medium for further elongation of the shoots and rooting. In 2 to 4 weeksthese shoots were further elongated and the roots were well developed.The rooted plants were hardened and established in green house.

All the tissue cultured plants grew normally till maturity. Seed settingwas normal in all the plants.

The calli produced from cut ends of the hypocotyls were subcultured atan interval of 2 to 4 weeks preferably 3 weeks interval on MS0Z₂ medium.On subculture these calli were grown in size.

6. A. 3. Mature Embryo

The seeds mentioned in 6.A. were used in these experiments; preferablythese seeds were imbibed in sterile water. The embryos were isolatedfrom these seeds in sterile conditions by pressing to remove the seedcoat with tweezers or any other means. The imbibed seeds are preferredover non-imbibed (However the positive responses were obtained fromimbibed and non-imbibed seeds). The cotyledons were separated fromembryos and these embryos measure 1 to 8 mm (preferably 5 mm) long atthe time of isolation. These isolated embryos were washed in sterilewater and blot dried. These embryos were wounded at the plumule tip.

These embryos were transferred on MS0Z2 medium for 2 to 4 weekspreferably for 3 weeks period. Multiple shoot bud clumps were developedand subcultured at 2 to 4 weeks interval preferably at 3 weeks intervalon MS0 medium. The frequency of shoot bud induction from the explantswas greater than 90% on MS0Z2 medium. Upon further transfer of themultiple shoot buds up to 5 fold multiplication was obtained. Shoots(preferably 1.5 cm) were transferred on MS0 medium in bottles forfurther elongation and rooting. After 1 to 5 weeks in the rooting mediumthe shoots were further elongated and the roots were well developed.

These (R0) regenerated plants were hardened and established in greenhouse. All the tissue cultured plants grew normally till maturity. Seedsetting was normal in all the plants.

6. B. Okra Transformations for Transgene Expression Preparation ofExplants for Transformation

6. B.1. Cotyledon with Petiole

Preparation of cotyledon explants is mentioned in 6. A.1.

6. B.2. Hypocotyl

Preparation of hypocotyl explants is mentioned in 6. A.2.

6. B.3. Mature Embryo

Preparation of mature embryo explants is mentioned in 6. A.3.

6. B. 4. Immature Embryo

Green fruits were collected (2 cm to 12 cm) and surface sterilized in0.1% (weight/volume) HgCl₂ in distilled water (for 1 to 60 minutespreferably for 5 minutes) and further washed in sterile distilled water.

Immature embryos were isolated from these fruits in sterile conditions(preferably by using tweezers or any other means) and used fortransformation.

6. B.5. Leaf Lamina

Leaf lamina were isolated from Okra plants (preferably from in vitrogrown) and used for transformation.

6. B.6. Cotyledonary Axil

The seeds were surface sterilized preferably in 0.1% (weight/volume)HgCl₂ in distilled water (for 1 to 60 minutes, preferably 30 minutes).These seeds were washed many times in sterile distilled water andinoculated on a medium (preferably on MS0 medium in bottles) andincubated under light for the germination of seeds. The explants viz.,cotyledon axils were excised from seedlings (5 to 20 days, preferably 12days old seedlings) and used for transformation.

6. B.7. Shoot Tip

The seeds were surface sterilized preferably in 0.1% (weight/volume)HgCl₂ in distilled water (for 1 to 60 minutes, preferably 30 minutes).These seeds were washed many times in sterile distilled water andinoculated on MS0 medium in bottles and incubated under light for thegermination of seeds. The explants viz., shoot tips were excised fromseedlings (preferably 12 days old seedlings) and used fortransformation.

6. B.8. Anther

Anthers were isolated in sterile conditions from unopened flower buds orflowers were used for the transformations.

6. B.9. Root

The roots were excised from plants (preferably grown in vitro) and wereused for transformation.

6. B.10. Callus

The calli grown in vitro, were used for transformations. These explants,as mentioned in above sections 6. B.1 to 6. B.10, were used forAgrobacterium-mediated method or Biolistic method of transformation. Thetransformed plant cells or tissues or plant were analyzed for thetransient/or stable GUS assay. The transformed plants were furtheranalyzed using standard molecular tools well known in prior art such asSouthern blotting, copy number estimation, and western blotting.

6. C. Agrobacterium-Mediated Method of Transformation

The vector pC 1301 contains the GUS and hpt genes linked to 35S promoterfrom Cauliflower Mosaic Virus (CaMV 35 S) and introduced intoAgrobacterium tumefaciens strain LBA 4404 to produce recombinant strain.

Recombinant A. tumefaciens (LBA 4404 pC 1301) strain was inoculated intoa suitable medium for the growth of Agrobacterium. Usually Agrobacteriumwas inoculated into 25 ml of sterile 2YT medium (pH 7) in a flask. 2YTmedium contains 1% Yeast extract, 1.6% Tryptone and 0.5% NaCl. Theantibiotics 10 mg/l Rifampicin, 20 mg/l Streptomycin, and 75 mg/lKanamycin were added before inoculating bacteria for the selectivegrowth of recombinant Agrobacterium with the plasmid 1301. Differentplasmids containing DNA/RNA sequence of interest were also used forproducing recombinant Agrobacterium strains for Example plasmid pC 1201can also be used for producing recombinant Agrobacterium strains Yeastextract, Tryptone and NaCl were purchased from HiMedia Labs, Mumbai,India. The bacteria were inoculated in 2YT medium in flask and kept on ashaker to get Optical Density (600 nm) of 0.01 to 2.5 preferably 1.8.

6. D. Inoculation of Explants with Recombinant Agrobacterium Tumefaciens

The above prepared explants (described in 6.B.1 to 6 B.10) wereinoculated in Agrobacterium suspension (preferably 15 minutes), blotteddry on sterile filter paper and later transferred to Petri dishes(preferably 20 embryos per Petri dish) on MSOAs medium [MSOAs mediumcontains MS salts (Murashige and Skoog. Physiol. Plant. (1962)15:473-497, B5 vitamins (Gamborg et al., Exp. Cell Res. (1968)50:151-158) 0.8% agar, 3% sucrose and pH 5.8 (Preferably enriched with3,5-Dimethoxy-4-hydroxyaceto-phenone, preferably in the concentration of100 mM).

These petri dishes were incubated in tissue culture incubation room.After the co-cultivation period (2 to 5 days, preferably 2 days) theseexplants were assayed for transient GUS expression or transferred ontoMS0Z₂H₁₀C medium MS0Z₂H₁₀C contains MS salts, B5 vitamins, 3% sucrose,0.8% agar, pH 5.8, cytokinin preferably Zeatin in the range 0.05 to 5mg/l, (preferably 2 mg/l Zeatin) supplemented with 5 mg/l to 100 mg/lHygromycin B (preferably 10 mg/l Hygromycin B) and taken for the stableGUS assay.

GUS activity was detected by the histochemical assay using5-bromo-4-chloro-3-indolyl-β-D-glucoronide as substrate (Jefferson etal., Proc. Natl. Acad. Sci. USA (1986) 83:8447-8451) and were incubatedin 100 mM phosphate buffer (pH 7.0) containing 1 mg/ml X-gluc forovernight at 37° C. temperature.

X-gluc was purchased from Duchefa, Haarlem, The Netherlands.

All the type of explants showed GUS expression. The results of these GUSassay are tabulated in 6. F as Table 1. The various explants showing GUSexpression are shown in the following figures. The mature embryo havingGUS expression is shown in FIG. 1A. The cotyledonary axil having GUSexpression is shown in FIG. 1B. The shoot tip having GUS expression isshown in FIG. 1C. The transformed callus having GUS expression is shownin FIG. 1D.

6. E. Microprojectile/Particle Bombardment-Method of Transformation

A number of plasmids were used in these experiments including plasmid pC1301 and plasmid ‘pC 1201’ for transformation.

All experiments were conducted with the Biolistic PDS-1000/He system(Sanford, TIB (1988) 6:299-302. Sanford et al., Technique J. MethodsCell Mol. Biol. (1991) 3:3-16) using tungsten or gold particles (3 mg)in the diameter of 0.1 to 3 μm. These particles were previously washedin ethanol were in aqueous suspension (50 μl) were coated with 5 to 10μg of plasmid DNA. The procedure followed for the coating of DNA on goldwas as described by Kikkert et al., Plant Cell Tissue and Organ Culture(1993) 33:221-226.

The particles were finely dispersed with an ultra sonicator (ElmaTransonic 460 Lab-Line Instruments Inc., IL, USA) before bombardment.

The explants were arranged in the center of the petri plate on a tissueculture medium. Bombardment pressure in the range of 900 to 1500 PSI(preferably 1100 PSI) was used. The distance from the launching platewas in the range of 6 to 18 cm, (preferably 6 cm) were used. (The goldand tungsten particles were purchased from Bio-Rad Labs, 2000 AlfredNobel Drive, Hercules, Calif. 94547 USA).

After bombardment of the explants as mentioned in section 6 E, thesewere placed on MS0 medium for 2 to 5 days (preferably 3 days). After 2to 5 days (preferably 3 days) these embryos were used for transient GUSassay or transferred on MS0Z₂H₁₀ medium [MS0Z₂H₁₀C contains MS salts, B5vitamins, 3% sucrose, 0.8% agar, pH 5.8, cytokinin preferably Zeatin inthe range 0.05 to 5 mg/l, (preferably 2 mg/l Zeatin) supplemented with 5mg/l to 100 mg/l Hygromycin B (preferably 10 mg/l Hygromycin B) andtaken for the stable GUS assay.

GUS activity was detected by the histo chemical assay using5-bromo-4-chloro-3-indolyl-β-D-glucoronide as substrate (Jefferson etal., Proc. Natl. Acad. Sci. USA (1986) 83:8447-8451) and were incubatedin 100 mM phosphate buffer (pH 7.0) containing 1 mg/ml X-gluc forovernight at 37° C. temperature.

All the types of explants showed GUS expression. Various explantsshowing GUS expression are shown in the following figures. Thetransformed mature embryo showing GUS activity is shown in FIG. 2A. Thetransformed callus showing GUS activity is shown in FIG. 2B and thetransformed anther showing GUS activity is shown in FIG. 2C.

The results of these GUS assay are tabulated in Table 1 and in sectionbelow.

6. F. Results of Transient GUS Expression Using Different Okra Explants

Various explants were used for transformation using bothAgrobacterium-mediated transformation and Biolistic-methods. Theexplants were screened using GUS assay. The data from this assay aretabulated in Table 1.

TABLE 1 Agrobacterium- Biolistic- mediated mediated transformationtransformation # of GUS explants # of GUS explants Sr. positive/# ofexplants positive/# of explants No. Explant assayed assayed 1 Cotyledonwith petiole 35/40 34/40 2 Hypocotyl 30/35 31/38 3 Mature embryo 38/3842/42 4 Immature embryo 36/37 24/40 5 Leaf lamina 37/37 17/40 6Cotyledonary axil 21/30 14/40 7 Shoot tip 25/30 16/29 8 Anther 22/4012/30 9 Root 09/40 12/40 10 Callus 09/40 08/40

The above mentioned explant types were also positive in stable GUSexpression in GUS assays when tested after 1 month. It is clear fromthese experiments that all explants used are amenable to transformation.However it is evident that mature embryo is the most efficient explantto use for transformation either by Agrobacterium-mediated orBiolistic-method. The various explant types were carried forward for thegeneration of the transgenic plants. The transformed plants were alsoanalyzed using standard molecular methodology known in the prior art.These plants were advanced to subsequent generations and analyzed forthe presence of heterologous genes integrated in genome. These areillustrated in the following examples.

The following examples are for understanding the invention and shouldnot be construed as to limit the scope of the invention.

EXAMPLES Example 1 Agrobacterium-Mediated Transformation of Okra EmbryosUsing GUS and hpt Genes (Marker Based/Linked System, Co-Cultivation onMS0 Medium)

A. Preparation of Plant Material

Okra genotypes representing a variety of agronomic varieties or hybrids[including Arka Anamika, Parbhani Kranti and proprietary lines ofMaharashtra Hybrid Seeds Company Ltd. (MHSCL)] were used in theseexperiments.

The seeds were surface sterilized preferably in 0.1% (weight/volume)HgCl₂ in distilled water (for 1 to 60 minutes, preferably 30 minutes).These mature seeds were washed many times in sterile distilled water.The surface sterilized seeds were imbibed in sterile water for a periodof 0 to 48 hours preferably for 16 hrs.

The embryos were isolated from these seeds in sterile conditions bypressing to remove the seed coat with tweezers or any other means. Thecotyledons were separated from embryos, and these embryos measure 1 to 8mm (preferably 5 mm) long at the time of isolation. These isolatedembryos were washed many times in sterile water blotted dry on filterpaper. These embryos were wounded at the plumule tip.

B. Preparation of Transgenic Agrobacterium Tumefaciens:

The vector pC 1301 contains the GUS and hpt genes linked to 35S promoterfrom Cauliflower Mosaic Virus (CaMV 35S) and introduced intoAgrobacterium tumefaciens strain LBA4404.

A. tumefaciens (LBA 4404 pC 1301) was inoculated into a suitable mediumfor the growth of Agrobacterium. Usually Agrobacterium was inoculatedinto 25 ml of sterile 2YT medium (pH 7) in a flask. 2YT medium contains1% Yeast extract, 1.6% Tryptone and 0.5% NaCl. The antibiotics 10 mg/lRifampicin, 20 mg/l Streptomycin, and 75 mg/l Kanamycin were addedbefore inoculating bacteria for the selective growth of Agrobacteriumwith the plasmid 1301.

Yeast extract, Tryptone and NaCl were purchased from HiMedia Labs,Mumbai, India. The bacteria were inoculated in 2YT medium in flask andkept on a shaker to get Optical Density (600 nm) of 0.01 to 2.5,preferably 1.8.

C. Inoculation of Explants with Recombinant Agrobacterium Tumefaciens

These wounded embryos were inoculated in Agrobacterium suspension(preferably 15 minutes), blotted dry on sterile filter paper and latertransferred to Petri dishes (preferably 20 embryos per petri dish) onMS0 medium [MS0 medium contains MS salts (Murashige and Skoog., Physiol.Plant. (1962) 15:473-497, B5 vitamins (Gamborg et al., Exp. Cell Res.(1968) 50:151-158) 0.8% agar, 3% sucrose and pH 5.8.

D. Regeneration of Transformed Plants after Co-Cultivation

The petri dishes containing wounded embryos were incubated in tissueculture incubation room. After co-cultivation period (2 to 5 dayspreferably 2 days of co-cultivation) these explants were washed inliquid MS0 medium (liquid MS0 medium contains MS salts, B5 vitamins, 3%sucrose and pH 5.8) with 500 mg/l Cefotaxime to kill the Agrobacteriumand transferred on MS0Z₂H₁₀C medium [MS0Z₂H₁₀C contains MS salts, B5vitamins, 3% sucrose, 0.8% agar, pH 5.8, cytokinin/s preferably Zeatinin the range 0.05 to 5 mg/l, (preferably 2 mg/l Zeatin) supplementedwith 5 mg/l to 100 mg/l Hygromycin B (preferably 10 mg/l Hygromycin B)were incubated per petri dish and were transferred onto fresh mediumafter a period of 2 to 5 weeks (preferably 3 weeks).

Between 4^(th) and 7^(th) weeks on MS0Z₂H₁₀C medium, multiple shoot budsdeveloped from the plumule of the embryo (FIG. 8).

These multiple shoot buds were subcultured on MS0H₁₀C (MS0H₁₀C containsMS salts, B5 vitamins, 3% sucrose, 0.8% agar and adjusted the pH 5.8supplemented with 10 mg/l Hygromycin B and 500 mg/l Cefotaxime) mediumat the interval of 2 to 4 weeks. These shoot bud clumps are shown inFIG. 8 were separated by cutting and subcultured on same media for 3weeks to develop shoots (preferably shoots of the size 1.5 cm). The saidshoots were transferred onto MS0H₁₀C medium in bottles for furtherelongation and rooting. After 1 to 5 weeks in the rooting medium, theshoots were elongated further and the roots were well developed (FIG.3B).

The rooted plants were hardened and established in green house (FIG.3C). All these plants grew normally till maturity. Seed setting wasnormal in all the plants.

A total of 180 embryos were inoculated in Agrobacterium in thisexperiment. Rooted plants were tested by GUS assay. A total of 6 plantswere positive for GUS expression.

Zeatin and agar were purchased from HiMedia Labs, Mumbai, India.Hygromycin B was purchased from A.G. Scientific, Inc. 6450 Lusk Blvd,San Diego, USA. 3,5-Dimethoxy-4-hydroxyaceto-phenone was purchased fromSigma-Aldrich Chemie Gmbh, Riedstr. Steinheim, Germany.

Sucrose was purchased from Sisco Research Lab. Pvt. Ltd., Mumbai, India.

E. Assay for Gus Expression

GUS activity was detected by the histochemical assay using5-bromo-4-chloro-3-indolyl-β-D-glucoronide as substrate (Jefferson etal., Proc. Natl. Acad. Sci. USA (1986) 83:8447-8451) and were incubatedin 100 mM phosphate buffer (pH 7.0) containing 1 mg/ml X-gluc forovernight at 37° C. temperature.

These 6 primary transformants (T₀ generation plants) were established ingreen house and were selfed and the T₁ generation seeds were collected.Two days old T₁ generation seedlings were used for GUS assay. The GUSexpression was found in the T₁ seedlings tested as shown in FIG. 4. Thesegregation pattern for GUS activity in these progenies indicates thatthe integration of GUS gene appears to be confined to a single locus inthe genome (Table No. 2).

Transgenic T₁ generation okra seedlings are positive for GUS expression

TABLE NO. 2 Segregation analysis of GUS gene expression in T₁ generationseedlings. Number of Number of Chi² value Number of T₁ seedlingspositive seedlings for the Segregation ratio seedlings tested for GUSnegative for GUS segregation of (Transgenic:Non Plant ID by GUS assayexpression expression GUS gene transgenic) TOG3 24 20 4 0.88 3:1

To rule out the possibility of GUS expression in non-transgenic plants,samples from these control plants were also assayed. The GUS expressionwas not found in these non-transgenic control plants. This resultconfirms that plants showing GUS activity are transgenic as shown inFIG. 4.

These plants were selfed and further the R₁ generation seeds werecollected. R₁ generation seedlings were raised from these seeds. Theleaf samples from these non transgenic plants were used as controls forthe GUS assay.

The GUS activity was not found in the non transgenic seedlings asexpected.

F. Negative Control for Transformation

Negative controls were maintained in each experiment to ensure that thehygromycin killed the growth of non-transgenic tissue. After theisolation, these embryos were wounded as mentioned above and withoutinoculating in bacteria, embryos were incubated for 2 days at the rateof 10 embryos per plate on MS0As medium. After 2 days, these embryoswere transferred on MS0Z₂H₁₀C for 2 to 4 weeks' period. All theseembryos completely bleached on selection medium by the end of 5^(th)subculture. This indicates that Hygromycin concentration (5-100 mg/l,preferably 10 mg/l) was sufficient to control the non-transgenic growthof tissue in these experiments.

Example No. 2 Agrobacterium-Mediated Transformation of Okra EmbryosUsing GUS and Hpt Genes (Marker Based System, Co-Cultivation on MS0Z₂ asMedium)

A. Preparation of Plant Material

Okra genotypes representing a variety of agronomic varieties or hybrids[including Arka Anamika, Parbhani Kranti and proprietary lines ofMaharashtra Hybrid Seeds Company Ltd. (MHSCL)] were used in theseexperiments.

The seeds were surface sterilized preferably in 0.1% (weight/volume)HgCl₂ in distilled water for 1 to 60 minutes (preferably 30 minutes).These seeds were washed many times in sterile distilled water andpreferably imbibed in sterile water.

The embryos were isolated from these seeds in sterile conditions bypressing to remove the seed coat with tweezers or any other means. Theimbibed seeds were preferred over non-imbibed. The cotyledons wereseparated from embryos, and these embryos measure 1 to 8 mm long(preferably 5 mm long) at the time of isolation. These isolated embryoswere washed many times in sterile water blotted dry on filter paper andplaced on soaked sterile filter paper. These embryos were wounded at theplumule tip.

B. Preparation of Transgenic Agrobacterium Tumefaciens:

The vector pC 1301 contains the GUS and hpt genes linked to 35S promoterfrom Cauliflower Mosaic Virus (CaMV 35S) and introduced intoAgrobacterium tumefaciens strain LBA4404.

The recombinant A. tumefaciens (LBA 4404 pC 1301) was inoculated into asuitable medium for the growth of Agrobacterium. Usually Agrobacteriumwas inoculated into 25 ml of sterile 2YT medium (pH 7) in a flask. 2YTmedium contains 1% Yeast extract, 1.6% Tryptone and 0.5% NaCl. Theantibiotics 10 mg/l Rifampicin, 20 mg/l Streptomycin, and 75 mg/lKanamycin were added before inoculating bacteria for the selectivegrowth of Agrobacterium with the plasmid 1301.

Yeast extract, Tryptone and NaCl were purchased from HiMedia Labs,Mumbai, India. The bacteria were inoculated in 2YT medium in flask andkept on a shaker to get Optical Density (600 nm) in the range of 0.01 to2, preferably 1.8.

C. Inoculation of Explants with Transgenic Agrobacterium Tumefaciens

These wounded embryos were inoculated in recombinant Agrobacteriumsuspension (preferably 15 minutes), blotted dry on sterile filter paperand later transferred to Petri dishes on MS0Z₂As medium (MS0Z₂Ascontains 0.05 to 5 mg/l Zeatin, preferably 2 mg/l Zeatin, MS salts(Murashige and Skoog., Physiol. Plant. (1962) 15:473-497), B5 vitamins(Gamborg et al., Exp. Cell Res. (1968) 50:151-158) 0.8% agar, 3% sucroseand pH 5.8 enriched with preferably 100 mM3,5-Dimethoxy-4-hydroxyaceto-phenone).

After the co-cultivation (2 to 5 days preferably 2 days ofco-cultivation), these explants were washed in liquid MS0 medium (liquidMS0 medium contains MS salts, B5 vitamins, 3% sucrose and pH 5.8) with500 mg/l Cefotaxime to kill the Agrobacterium and were transferred onMS0Z2H10C medium (MS0Z2H10C contains MS salts, B5 vitamins, 3% sucrose,0.8% agar, pH 5.8, 0.05 to 5 mg/l Zeatin (preferably 2 mg/l Zeatin)supplemented with 5 mg/l to 100 mg/l Hygromycin B (preferably 10 mg/lHygromycin B) and preferably 500 mg/l Cefotaxime (any other antibiotic)to kill the Agrobacterium. About 5 to 20 developing embryos (preferably10 embryos) were incubated per petri dish and were transferred ontofresh medium after a period of 2 to 5 weeks (preferably 3 weeks).

By the end of 4th to 7th weeks, preferably 6th week on MS0Z2H10C medium,multiple shoot buds developed from the plumule tip of the embryo.

These multiple shoot buds were subcultured on MS0H10C (MS0H10C containsMS salts, B5 vitamins, 3% sucrose, 0.8% agar and adjusted the pH 5.8supplemented with 10 mg/l Hygromycin B and 500 mg/l Cefotaxime) mediumat the interval of 3 weeks. These shoot bud clumps were separated bycutting and subcultured on same media for 2 to 5 weeks to develop shoots(preferably about 1.5 cm in size). The said shoots were transferred ontoMS0H10C medium in bottles for further elongation and rooting. After 2 to4 weeks in the rooting medium, the shoots were elongated further and theroots were well developed.

The rooted plants were hardened and established in green house. A totalof 300 embryos were inoculated in Agrobacterium in this experiment.Rooted plants were tested by GUS assay. A total of 6 plants werepositive for GUS expression. All these 6 plants grew normally tillmaturity. Seed setting was normal in all the plants. Experiments wereconducted several times to obtain transformed plants with similarresults.

GUS activity was detected by the histochemical assay using5-bromo-4-chloro-3-indolyl-β-D-glucoronide as substrate (Jefferson etal., Proc. Natl. Acad. Sci. USA (1986) 83:8447-8451). Plant segments orleaf segments were incubated in 100 mM phosphate buffer (pH 7.0)containing 1 mg/ml X-gluc overnight at about 370C temperature.

Zeatin and agar were purchased from HiMedia Labs, Mumbai, India

Hygromycin B was purchased from A.G. Scientific, Inc. 6450 Lusk Blvd,San Diego, USA.

3,5-Dimethoxy-4-hydroxyaceto-phenone was purchased from Sigma-AldrichChemie Gmbh, Riedstr. Steinheim, Germany.

D. Assay for GUS Expression

The leaves cut from rooted plants were taken for GUS assay. In thisexperiment 6 plants were positive for GUS expression.

To rule out the possibility of GUS expression in non-transgenic plants,samples from these control plants were also assayed. The GUS expressionwas not found in these non-transgenic control plants as expected. Thisresult confirms that plants expressing GUS gene were transgenic.

These primary transformants (T₀ generation plants) were established ingreen house and were selfed and the T₁ generation seeds were collected.These plants were advanced to further generations.

E. Negative Control for Transformation

Negative controls were maintained in each experiment to ensure that thehygromycin killed the growth of non-transgenic tissue. A total of 20embryos were maintained as negative control. After the isolation, theseembryos were wounded as mentioned above and without inoculating inbacteria, embryos were incubated for 2 days at the rate of 5 to 20,preferably 10 embryos per plate on MS0Z₂As medium. After 2 days, theseembryos were transferred on MS0Z₂H₁₀C for 2 to 5 weeks period. Out of 20embryos, 10 produced multiple shoot buds which were sub-cultured at 3weeks interval on MS0Z₂H₁₀C. But these multiple shoot buds werecompletely bleached by the end of 5^(th) subculture. This provesHygromycin concentration (5 to 100 mg/l, preferably 10 mg/l) wassufficient to control the non-transgenic growth of tissue in theseexperiments.

Example No. 3 Agrobacterium-Mediated Transformation of Okra EmbryosUsing Cry1A(c)/Cry2Ab etc. and NPT II Genes for the Generation of InsectResistance Transgenic Okra (Marker Based System, Cocultivation on MS0AsMedium)

A. Preparation of Explants:

Okra genotypes representing a variety of agronomic varieties or hybrids[including Arka Anamika, Parbhani Kranti and proprietary lines ofMaharashtra Hybrid Seeds Company Ltd. (MHSCL)] were used in theseexperiments.

The mature seeds were surface sterilized preferably in 0.1%(weight/volume) HgCl₂ in distilled water for 1 to 60 minutes (preferably30 minutes). These seeds were washed many times in sterile distilledwater (Preferably, these seeds were imbibed in sterile water).

The embryos were isolated from these seeds in sterile conditions bypressing to remove the seed coat with tweezers or any other means. Theimbibed seeds were preferred over non-imbibed. However the non-imbibedseeds also can be used. The cotyledons were separated from embryos, andthese embryos measure 1 to 8 mm preferably 5 mm long at the time ofisolation. These isolated embryos were washed many times in sterilewater blotted dry on filter paper and placed on soaked sterile filterpaper. These embryos were wounded at the plumule tip and used for theco-cultivation in Agrobacterium suspension.

B. Preparation of Recombinant Agrobacterium Tumefaciens

The vectors used were pC 2300 which carries Cry2Ab/Cry1A(c) gene andnptII gene as plant selectable marker in the T-DNA of this plasmid. Oneof these plasmids was introduced into the Agrobacterium tumefaciensstrain EHA 105 and used for the transformations.

The recombinant Agrobacterium strain carrying Bt gene (Cry2Ab orCry1A(c) etc.) in the plasmid pC2300 was used for transformation of okraplants.

The antibiotic 50 mg/l kanamycin and 10 mg/l chloramphenicol were addedto 2YT medium for the selective growth of the Agrobacterium with theplasmid containing either Cry1A(c) or Cry2Ab gene.

The antibiotic kanamycin (preferably 50 mg/l) was used to select thetransgenic tissue on MS0Z₂K₅₀C medium (MS0Z₂K₅₀C medium contains MSsalts, B5 vitamins, 3% sucrose, 0.8% agar, pH 5.8, 2 mg/l Zeatinsupplemented with 25 to 200 mg/l, preferably 50 mg/l kanamycin andpreferably 500 mg/l Cefotaxime or any other antibiotic to kill theAgrobacterium). Kanamycin was purchased from Macleods Pharma., Daman,U.T., India.

C. Analysis of Putative Transgenic Plants Using Double Antibody SandwichELISA.

The putative transformed plants were tested for the expression of the Btgenes, Cry1A(c) or Cry2Ab, using ELISA assay.

The ELISA plate was coated with monoclonal antibodies specific toCry1A(c) or Cry2Ab protein and these plates were supplied from DesigenDiagnostics, Division of MAHYCO seeds Ltd., Maharashtra, India.According to the manufacturer's protocol the assay was carried out asfollows. 2 to 4 leaf discs of the diameter 1 cm were taken from putativetransgenic plants and control plants. These leaf samples were extractedin 500 μA of 1×PBST buffer. 50 μl of sample was loaded to each well inthe pre-coated plate. After sample loading 15 μl of polyclonalantibodies specific to Cry1A(c) or Cry2Ab in the ratio 1:20,000 dilutionin PBSTO was added to each well. This plate was stored at 4° C.overnight. The overnight incubated plate was washed thrice with PBST onthe next day. 200 μl per well detection antibody labeled with alkalinephosphate at 1:6000 dilution in PBSTO, was added to these washed plates.This plate was incubated for 2 hours at room temperature. After theincubation this plate was washed thrice with PBST. Finally 250 μlsubstrate buffer containing 1 mg/ml paranitro phenyl phosphate was addedper well and the color development was recorded at 405 nm wavelengthusing an ELISA reader. The positive samples were selected on thedevelopment of yellow color giving OD value >0.2 (after 30 minutesincubation) after subtracting the blank value which was compared to thenegative (non-transgenic) control. Composition of all the buffers usedfor ELISA is summarized in Table 3.

TABLE NO. 3 Details for the buffers used for ELISA for screeningputative transgenic plants Buffer Components for 1 liter Make 10X 10XPBST buffer was prepared by Sodium chloride was pur- PBST adding 80 gm/lSodium chloride, chased from Merck (India) (pH 7.4) 11.5 gm/l Sodiumphosphate Limited, Worli, Bombay, dibasic, 2 gm/l potassium chlorideIndia. Sodium phosphate and 2 gm/l Potassium dihydrogen dibasic waspurchased phosphate and adjusted the pH to from Sisco Research Lab. 7.4.After making up the volume to Mumbai, India. Potassium 1 liter, 5 ml ofTween 20 was chloride, Potassium added. dihydrogen phosphate werepurchased from HiMedia Labs, Mumbai, India. 1X PBST 1X PBST buffer wasprepared by diluting 10X PBST buffer using distilled water 1X 1X PBSTwith 0.5% Ovalbumin Ovalbumin was pur- PBSTO chased from HiMedia Labs,Mumbai, India. Substrate Ethanolamine = 96 ml Ethanolamine was pur-buffer HCl + Milli Q water = 52 m1 + 48 chased from SD ml = 100 ml fineChem. Boisar, India. Made up the final volume to 1 liter by adding MilliQ waterELISA results of 25 transgenic Okra plants positive for Bt with therespective ELISA readings.

TABLE NO. 4 Sr. No. Plant ID ELISA reading 1 TOC2b 0.64 2 TOC3a 0.74 3TOC4a 0.26 4 TOC5 0.41 5 TOC9a 0.64 6 TOC12 0.66 7 TOC13 0.36 8 TOC29b0.45 9 TOC32a 0.58 10 TOC41a 0.68 11 TOC42a 0.58 12 TOC43a 0.79 13TOC44a 0.81 14 TOC45 0.64 15 TOC46a 0.6 16 TOC49a 0.63 17 TOC50a 0.62 18TOC52a 0.75 19 TOC53 0.78 20 TOC54 0.7 21 TOC55a 0.57 22 TOC58 0.64 23TOC59 0.32 24 TOC60a 0.44 25 TOC62a 0.49 Okra (Non NTO1 0.01 transgenic)D. Insect Bioassays Using Transgenic and Nontransgenic Fruits

The larvae (second instar) of these insects pests viz., Shoot and fruitborer (Earias vitella, E. insulana) and the Fruit borer (Helicoverpaarmigera) were reared in the laboratory, were released separately on theyoung fruits from these transgenic plants. The fruits were placed inglass bottle and covered with cloth for aeration. Larval mortality ifany was recorded after 3 days. On transgenic fruits all the larvae ofthese insects were dead where as on non transgenic they were alive andhealthy. These results indicate very high resistance to the target pestsin the transgenic plants (Data of bioassay is summarized in Table No.5).

TABLE NO. 5 Results of insect bioassays using Okra fruit samples Numberof Status of larvae Plant type Name of insect larvae used larvae/fruitafter 3 days Transgnic Bt Okra Helicoverpa armigera 5 Dead Nontransgenic Helicoverpa armigera 5 Live control Okra Transgnic Bt OkraEarias vitella 5 Dead Non transgenic Earias vitella 5 Live control Okra

Fruits from non transgenic plants were used in the insect bioassays asmentioned above in Table No. 5. The fruits of these plants weresusceptible for the insect pests damage.

Mendelian segregation ratio of 3:1, for presence the of Btprotein:absence of Bt protein, was observed in the progenies of a largenumber of transgenic lines, whereas few lines did not segregate in 3:1ratio. The segregation results of 4 transgenic lines are tabulated inthe Table No. 6.

TABLE NO. 6 Segregation analysis of Bt gene in transgenic Okra T₁generation seedlings from four events. Number of Number of Number ofseedlings seedlings Chi² value for Segregation T₁ seedlings positive fornegative for the segregation ratio Sr. No. Plant ID tested by ELISA Btin ELISA Bt in ELISA of Bt gene (Bt:Non Bt) 1 TOC14a 28 12 16 15.42 3:42 TOC17a 26 16 10 2.52 3:1 3 TOC24A 17 11 6 0.96 3:1 4 TOC38 17 13 40.02 3:1E. Tissue Culture Controls

The non transgenic (tissue cultured) control plants were selfed and theR1 generation seeds were collected. R1 generation seedlings were raisedfrom these seeds. The leaf samples from these non transgenic plants wereused as controls for the ELISA. The Bt protein activity was not found inthe non transgenic seedlings as expected.

F. Negative Control for Transformation

Negative controls were maintained in each experiment to ensure that thekanamycin killed the growth of non-transgenic tissue. After theisolation, these embryos were wounded as mentioned above and withoutinoculating in bacteria, these embryos were incubated for 2 days at therate of 10 embryos per plate on MSOAs medium. After 2 days, theseembryos were transferred on MS0Z₂K₅₀C for a period of 3 weeks ontoMS0Z₂K₅₀C medium.

These explants bleached on 1^(st) selection in the period of 3 weeks.This proves kanamycin concentration (preferably 50 mg/l) was sufficientto kill the non-transgenic tissue in these experiments.

Example No. 4 Agrobacterium (EHA 105 Strain)-Mediated Transformation ofOkra Embryos Using Cry1A(c) and NPT II Genes for the Generation ofInsect Resistant Transgenic Okra [Marker Based System, Cocultivation onMS0 Medium]

A. Preparation of Plant Material

Okra genotypes representing a variety of agronomic varieties or hybrids[including Arka Anamika, Parbhani Kranti and proprietary lines ofMaharashtra Hybrid Seeds Company Ltd. (MHSCL)] were used in theseexperiments.

The seeds were surface sterilized preferably in 0.1% (weight/volume)HgCl2 in distilled water for 1 to 60 minutes (preferably 30 minutes).These seeds were washed many times in sterile distilled water and thenimbibed in sterile water for 0 to 48 hours preferably 16 hrs.

The embryos were isolated from these seeds in sterile conditions bypressing to remove the seed coat with tweezers or any other means. Thecotyledons were separated from embryos, and these embryos measure 1 to 8mm preferably 5 mm long at the time of isolation. These isolated embryoswere washed many times in sterile water blotted dry on filter paper andplaced on soaked sterile filter paper. These embryos were wounded at theplumule tip.

B. Preparation of Transgenic Agrobacterium Tumefaciens

The vectors used were pC 2300 which carries either Cry2Ab gene orCry1A(c) gene and nptII gene as plant selectable marker in the T-DNA ofthese plasmids. One of these plasmids was introduced into theAgrobacterium tumefaciens strain EHA 105 and used for thetransformations. The antibiotic 25 mg/l kanamycin and 10 mg/lchloramphenicol were added to 2YT medium for the selective growth of theAgrobacterium with the plasmid containing Cry1A(c) or Cry2Ab gene.

The Agrobacterium carrying either Cry2Ab or Cry1A(c) gene was used inthe transformations.

C. Inoculation of Explants with Recombinant Agrobacterium Tumefaciens

These wounded embryos were inoculated in Agrobacterium suspension(preferably 15 minutes), blotted dry on sterile filter paper and latertransferred in petri dishes (preferably 20 embryos per petri dish) on[MS0 medium contains MS salts (Murashige and skoog., Physiol. Plant.(1962) 15:473-497, B5 vitamins (Gamborg et al., Exp. Cell Res. (1968)50:151-158) 0.8% agar, 3% sucrose and pH 5.8].

After 2 to 4 days of co-cultivation (preferably 2 days ofco-cultivation) these explants were washed in liquid MS0 medium (liquidMS0 medium contains MS salts, B5 vitamins, 3% sucrose and pH 5.8) with500 mg/l Cefotaxime to kill the Agrobacterium and were transferred onMS0Z₂K₅₀C medium (MS0Z₂K₅₀C contains MS salts, B5 vitamins, 3% sucrose,0.8% agar, pH 5.8, 0.05 to 5 mg/l Zeatin (preferably 2 mg/l Zeatin)supplemented with Kanamycin preferably 50 mg/l Kanamycin) and preferably500 mg/l Cefotaxime (any other antibiotic) to kill the Agrobacterium). 5to 20 developing embryos (preferably 10 embryos) were incubated perpetri dish and were transferred onto fresh medium after a period of 2 to5 weeks (preferably 3 weeks).

In a period of 3 to 7 weeks on MS0Z₂K₅₀C medium, multiple shoot budsdeveloped from the plumule of the embryo.

These multiple shoot buds were sub cultured on MS0K₅₀C (MS0K₅₀C containsMS salts, B5 vitamins, 3% sucrose, 0.8% agar and adjusted the pH 5.8supplemented with 50 mg/l Kanamycin and 500 mg/l Cefotaxime) medium atthe interval of 3 weeks. These shoot bud clumps were separated bycutting and subcultured on same media for 2 to 5 weeks to develop shootsof size 0.5 cm to 5 cm preferably 1.5 cm. The said shoots weretransferred onto MS0K₅₀C medium in bottles for further elongation androoting. After 2 to 4 weeks in the rooting medium, the shoots wereelongated further and the roots were well developed.

The rooted plants were hardened and established in green house. Rootedplants were tested by ELISA. Three transgenic plants were generated from175 explants used in this transformation experiment.

Example No. 5 Agrobacterium-Mediated Transformation of CotyledonExplants Using Cry1A(c) and NPT II Genes for the Generation of InsectResistant Transgenic Okra (Marker Based System, Cocultivation on MS0Z₂AsMedium)

Okra genotypes representing a variety of agronomic varieties or hybrids[including Arka Anamika, Parbhani Kranti and proprietary lines ofMaharashtra Hybrid Seeds Company Ltd. (MHSCL)] were used in theseexperiments.

The seeds were surface sterilized preferably in 0.1% (weight/volume)HgCl₂ in distilled water for 1 to 60 minutes (preferably 30 minutes).These seeds were washed many times in sterile distilled water.

The surface sterilized seeds were inoculated on MS0 medium in bottlesand incubated under light for the germination of seeds. The cotyledonwith petiole explants were excised from seedlings (1 to 20 days oldseedlings preferably from 12 days old seedlings). These explants wereinoculated in the Agrobacterium strain EHA 105 with the vector pC 2300which carries Cry1A(c) gene and nptII gene. These explants wereinoculated in Agrobacterium suspension (preferably 15 minutes) andtransferred on MS0Z₂As medium as mentioned in Example. 3. After 2 to 4days (preferably 2 days) of co-cultivation these explants weretransferred on MS0Z₂K₅₀C medium.

From the cut ends of the petiole of these cotyledon explants multipleshoot buds and callus developed. The shoot buds produced from thepetiole of these explants were separated and subcultured on MS0K₅₀Cmedium for the further elongation of the shoots and rooting. Shootselongated and roots were well developed in rooting medium during aperiod of 2 to 4 weeks. The rooted plants were hardened and establishedin green house. These plants were tested by ELISA for the presence ofCry1A(c) protein as in Example. 3. The plants were negative for thepresence of Cry1A(c) protein. The calli produced from cut ends of thepetiole of these explants were subcultured at an interval of 2 to 5weeks on MS0Z₂KC medium. On subculture these calli were grown in size.The pieces of calli were tested by ELISA as mentioned in Example 3 forthe presence of Cry1A(c) protein. Out of 7 calli tested 4 were positivefor the presence of Cry1A(c) protein, which proves that these calli weretransgenic.

Negative Control for Transformation

Negative controls were maintained in each experiment to ensure that thekanamycin killed the growth of non transgenic growth. Cotyledon withpetiole were excised as mentioned in 3. A. 1 and with out inoculating inbacteria, were incubated on MS0Z₂As medium for 2 days at the rate ofpreferably 10 embryos per plate. After 2 days, these were transferred onMS0Z₂K₅₀C for 3 weeks period and sub-cultured at 3 weeks interval onMS0Z₂K₅₀C. But most of the multiple shoot buds bleached by the end of5^(t1) subculture. This proves kanamycin concentration preferably 50mg/l was sufficient to control the non-transgenic growth of tissue tosome extent in these experiments.

Example No. 6 Agrobacterium-Mediated, Co-Transformation of Embryos Usinga Plasmid (T-DNA) Carrying Cry1A(c)/Cry2AB etc. and Another Plasmid(T-DNA) Carrying NPT II and GUS Genes, for the Generation of Marker FreeBt Okra (Cocultivation on MS0As Medium) Agrobacterium Tumefaciens andthe Constructs Used

The method used was Agrobacterium-mediated, co-transformation, i.e. asingle Agrobacterium tumefaciens strain carrying two plasmids. TheAgrobacterium strain used was LBA 4404, carrying plasmid MH 0102 or MH0103 and pC 2301. The plasmid MH 0102 or MH 0103 and pC 2201 carryCry1A(c) or Cry2Ab and GUS & NPT II genes respectively.

The antibiotic 25 mg/l kanamycin and 10 mg/l chloramphenicol were addedto 2YT medium for the selective growth of the Agrobacterium LBA4404 withthe plasmids MH 0102 or MH 0103 and pC 2201. The tissue culture stepsfor the recovery of transgenic plants were as mentioned in Example 1.

A. Preparation of Plant Material

Okra genotypes representing a variety of agronomic varieties or hybrids[including Arka Anamika, Parbhani Kranti and proprietary lines ofMaharashtra Hybrid Seeds Company Ltd. (MHSCL)] were used in theseexperiments.

The preparation of plant material and inoculation of explants withAgrobacterium, and the tissue culture steps for the recovery of plantswere as mentioned in Example 3.

These putative transgenic plants were screened by GUS assay (for thepresence of marker gene) and ELISA (for the detection of Cry1A(c) orCry2Ab protein). A total of 10 plants were positive for GUS expression(Table 7). The transgenic plants were hardened and established in greenhouse.

GUS activity was detected by the histochemical assay using5-bromo-4-chloro-3-indolyl-β-D-glucoronide as substrate (Jefferson etal., Proc. Natl. Acad. Sci. USA (1986) 83:8447-8451) and were incubatedin 100 mM phosphate buffer (pH 7.0) containing 1 mg/ml X-gluc forovernight at 37° C. temperature.

Analysis of putative transgenic plants using double antibody sandwichELISA was performed as per the manufacturer's instruction as mentionedin Example No 3.

TABLE NO. 7 ELISA (Bt) and GUS assay results of 10 transgenic T₀generation plants generated from marker free system Sr. No Plant IDELISA reading GUS result 1 OCM1 0.531 + 2 OCM2a 0.736 + 3 OCM3 0.725 + 4OCM4 0.560 + 5 OCM5a 0.001 + 6 OCM10a 0.011 + 7 OCM11a 0.000 + 8 OCM12a0.001 + 9 OCM13a 0.002 + 10 OCM14a 0.000 + 11 (Okra NTO1 0.010 − Nontransgenic)

TABLE NO. 8 Segregation of marker and Bt genes in T₁ generation plantsderived from T₀ line No. OCM1) Sr. No. Plant ID ELISA reading GUS result1 OCM1-1 0.531 + 2 OCM1-2 0.675 + 3 OCM1-3 0.498 + 4 OCM1-4 0.656 + 5OCM1-5 0.000 + 6 OCM1-6 0.453 − 7 OCM1-7 0.687 +

The marker-free transgenic plants i.e. carrying Bt gene [Cry1A(c) orCry2Ab gene] but not the marker gene were identified in the followinggenerations from the segregated progenies, as indicated in Table No 8.

The seeds obtained from the marker-free transgenic plants andappropriate controls were further inoculated in vitro on MS basal mediumwith kanamycin 50 mg/l (MS0K₅₀ medium) to reconfirm the marker freestatus of these lines. These markers-free seedlings got bleached in 3weeks period. Similar results were also seen for non transgeniccontrols. However seedlings carrying marker gene (GUS positive)continued to grow on the medium with kanamycin 50 mg/l. These resultsreconfirm the marker free status of these lines.

Example No. 7 Biolistic Method of Transformation of Okra Plants

A. Preparation of Plant Material

Okra (Abelmoschus esculentus) genotypes representing a variety ofagronomic varieties or hybrids [including Arka Anamika, Parbhani Krantiand proprietary lines of Maharashtra Hybrid Seeds Company Ltd. (MHSCL)]were used in these experiments.

The seeds were surface sterilized preferably in 0.1% (weight/volume)HgCl₂ in distilled water (for 1 to 60 minutes, preferably 30 minutes).These mature seeds were washed many times in sterile distilled water.(Preferably, these surface sterilized seeds were imbibed in sterilewater).

The embryos were isolated from these seeds in sterile conditions bypressing to remove the seed coat with tweezers or any other means. Thecotyledons were separated from embryos and these embryos measure 1 to 8mm (preferably 5 mm) long at the time of isolation. These isolatedembryos were washed many times in sterile water blotted dry on filterpaper. These embryos were wounded at the plumule tip.

B. Particle Bombardment of Explants Using Biolistic System.

All experiments were conducted with the Biolistic PDS-1000/He system(Sanford, TIB (1988) 6:299-302. Sanford et al., Technique J. MethodsCell Mol. Biol. (1991) 3:3-16) using tungsten or gold particles (3 mg)in the diameter of 0.1 to 3 μm. These particles were previously washedin ethanol were in aqueous suspension (50 μl) were coated with 5 to 10μg of plasmid DNA. The procedure followed for the coating of DNA on goldwas as described by Kikkert et al., Plant Cell Tissue and Organ Culture(1993) 33:221-226.

The particles were finely dispersed with an ultra sonicator (ElmaTransonic 460 Lab-Line Instruments Inc., IL, USA) before bombardment.

The explants were arranged in the center of the petri plate on a tissueculture medium. Bombardment pressure in the range of 900 to 1500 PSI(preferably 1100 PSI) was used. The distance from the launching platewas in the range of 6 to 18 cm, (preferably 6 cm) were used. Variousexplants were used for particle bombardment. (The gold and tungstenparticles were purchased from Bio-Rad Labs, 2000 Alfred Nobel Drive,Hercules, Calif. 94547 USA).

After bombardment, the explants were placed on MS0 medium for 2 to 5days (preferably 3 days). After 2 to 5 days (preferably 3 days) theseembryos were used for transient GUS assay or transferred on MS0Z₂H₁₀medium [MS0Z₂H₁₀C contains MS salts, B5 vitamins, 3% sucrose, 0.8% agar,pH 5.8, cytokinin preferably Zeatin in the range 0.05 to 5 mg/l,(preferably 2 mg/l Zeatin) supplemented with 5 mg/l to 100 mg/lHygromycin B (preferably 10 mg/l Hygromycin B) and taken for the stableGUS assay.

C. GUS Assay of the Transformed Explants

GUS activity was detected by the histochemical assay using5-bromo-4-chloro-3-indolyl-β-D-glucoronide as substrate (Jefferson etal., Proc. Natl. Acad. Sci. USA (1986) 83:8447-8451) and were incubatedin 100 mM phosphate buffer (pH 7.0) containing 1 mg/ml X-gluc forovernight at 37° C. temperature.

All the types of explants showed GUS expression

Advantages

-   -   1. This invention provides plant regeneration from a variety of        explants of Okra or Abelmoschus species.    -   2. The shoot bud regeneration frequency from plumule of embryo        explants of Okra or Abelmoschus sps. is more than 80%.    -   3. This invention also provides methods for the further        multiplication of the shoot buds.    -   4. This invention also provides methods for the transient        expression of the transgene in high frequency using        Agrobacterium-mediated transformation.    -   5. This invention also provides methods for the transient        expression of the transgene in high frequency using        biolistic-mediated transformation.    -   6. This invention also provides methods for the        Agrobacterium-mediated genetic transformation of Okra or        Abelmoschus species for the first time.    -   7. This invention also provides methods for the        biolistic-mediated genetic transformation of Okra or Abelmoschus        species for the first time.    -   8. This invention also provides methods for the generation of        transgenic Okra or Abelmoschus sps with nucleotide sequences on        interest.    -   9. This invention also provides methods for the generation of        transgenic insect resistant Okra or Abelmoschus species.    -   10. This invention also provides methods for the generation of        marker-free insect resistant transgenic Okra or Abelmoschus        species.    -   11. This invention also provides methods for the generation of        marker-free transgenic Okra or Abelmoschus sps, with the DNA        sequences of interest.

REFERENCES

-   1. Herberlandt Sber. Akad. Wiss. Wien. (1902) 111:69-92).-   2. Chilton, Scientific American (1983) 248.6:36-45).-   3. Mangat and Roy., Plant Science (1986) 47:57-62.-   4. Roy and Mangat (Roy et al., Plant Science (1989) 60:77-82)-   5. Murai et al., Science (1983) 222:476-482.-   6. Fraley et al., Proc. Natl. Acad. Sci. USA (1983) 80:4803-4807-   7. Lorz et al., Mol. Gen. Genet., (1985) 199:178-182.-   8. Portrykus et al., Mol. Gen. Genet., (1985) 199:183-188;-   9. Crossway et al., Mol. Gen. Genet., (1986) 202:179-185;-   10. Klein et al., Nature (1987) 327:70-73-   11. Fromm et al., Proc. Natl. Acad. Sci. USA (1985) 82:5824-5828,-   12. Fromm et al., Nature (1986) 319:791-793.-   13. Murai et al., Science (1983) 222:476-482.-   14. Fraley et al., Proc. Natl. Acad. Sci. USA (1983) 80:4803-4807.-   15. De Block et al., The EMBO Journal (1984) 3:1681-1689.-   16. Horsch et al., Science (1985) 227:1229-1231.-   17. Klein et al., Nature (1987) 327:70-73.-   18. Klein et al., Bio/Technology (1992) 10:286-292.-   19. Casas et al., Proc. Natl. Acad. Sci. USA (1993) 90: 11212-11216.-   20. Yoder et al., Bio/Technology (1994) 12:263-267).-   21. Komori et al., The Plant Journal (1996) 10: 165-174-   22. Fischhoff et al., Bio/Technology (1987) 5:807-813.-   23. Johnson et al., PNAS (1990) 86:9871-9875.-   24. Murashige et al., Physiol. Plant. (1962) 15:473-497-   25. Gamborg et al., Exp. Cell Res. (1968) 50:151-158).-   26. Sanford, TIB (1988) 6:299-302.-   27. Sanford et al., Technique J. Methods Cell Mol. Biol. (1991)    3:3-16).-   28. Kikkert et al., Plant Cell Tissue and Organ Culture (1993)    33:221-226.-   29. Jefferson et al., Proc. Natl. Acad. Sci. USA (1986)    83:8447-8451)

We claim:
 1. A method of regeneration of okra plant, wherein said methodcomprising: (a) obtaining an explant from either seeds, seedlings orwhole plant, wherein the explants are selected from a group consistingof cotyledon with petiole, hypocotyl, Embryo, immature embryo, anther,and root, wherein the embryo is wounded at the plumule tip; (b)culturing said explant on a growth medium containing zeatin and vitaminsto obtain multiple shoot buds, wherein the growth medium does notcontain auxin; (c) culturing the shoot buds on a rooting medium toobtain rooted plantlets; and (d) transferring said rooted plantlets insoil, to obtain regenerated plants.
 2. The method as claimed in claim 1,wherein said seeds, seedlings or whole plant are selected from differentvarieties or hybrids of Abelmoschus esculentus.
 3. The method as claimedin claim 1, wherein the growth medium contains zeatin in the range of0.01 to 5 mg/l.
 4. The method as claimed in claim 1, wherein the rootingmedium is selected from a group consisting of MS medium containingzeatin, BAP, kinetin, and Adenine, MS medium containing zeatin, BAP,kinetin, and Adenine in combination and a basal MS medium.
 5. The methodas claimed in claim 1, wherein the rooting medium is a basal MS medium.6. An Abelmoschus esculantus or Okra plant produced by the method ofclaim 1.